We have reported that cultured mast (RBL-2H3) cells generate growth-promoting cytokines constitutively (e.g. TGFbeta) and inflammatory cytokines (e.g., TNFalpha and IL6) when stimulated. Production of inflammatory cytokines is mediated by mobilization of Ca2+, activation of protein kinase C (PKC), and a third synergising signal, tentatively identified as the p42 MAP kinase (ERK2) pathway (see last years report). Secretion of these cytokines is dependent on Golgi, mobilization of Ca2+, and activation of PKC and is readily blocked by selective disruption of any one of these three processes by pharmacologic agents. We have now positively identified the ERK2 pathway as a necessary signal for production of TNFalpha as follows. Of all stimulants tested, antigen is by far the most potent stimulant of this pathway and TNFalpha production. Both are inhibited equally well by dexamethasone, the kinase inhibitor, quercetin, and the MEK inhibitor, PD098059, at concentrations that have minimal effects on other stimulatory signals in RBL-2H3 cells. The small amounts of TNFalpha that are formed are secreted, consistent with previous indications that the MAP kinase pathway regulates production but not secretion of TNFalpha. Production of TNFalpha, however, is not affected by wortmannin, which inhibits phosphoinositide 3- kinase and, as a consequence, various other kinases including the JNK family of MAP kinases. In contrast to TNFalpha, the constitutive production of TGFbeta is unaffected by inhibitors of the MAP kinases and PKC, although a small additional increase in production TGFbeta can be elicited by antigen and this increase is suppressed by such inhibitors to indicate regulation of this inducible component of TGFbeta production. Thus, both constitutive and inducible pathways exist for production of cytokines and the inducible pathways can be selectively suppressed by pharmacologic agents. The studies with the above inhibitors reveal parallel effects on the phosphorylation of a cytosolic phospholipase A2 (cPLA2) by ERK2 and release of archidonic acid. These and previous studies permit the following conclusions. The ERK pathway, which is activated through PKC, calcium, and a Shc/Ras pathway that provides sustained signals for activation in antigen-stimulated cells, is required for phosphorylation and activation of cPLA. In antigen-stimulated cells, PKC causes transient stimulation of cPLA and arachidonic acid release, via the ERK pathway. Inhibitors of PKC thereby delay, but do not block, release of arachidonic acid whereas inhibitors of the ERK pathway suppress this release. Our studies provide for the first time plausible explanations for the actions of several experimental and therapeutic agents on release of inflammatory mediators from mast cells.